Substituted alkyl esters quinoxaline-di-N-oxide-2-carboxylic acid as growth promoting agents

ABSTRACT

Novel alkyl esters of quinoxaline-di-N-oxide-2-carboxylic acid substituted on the alkyl portion of the ester by hydroxy, acyloxy, N-alkyl carbamyloxy, dialkylaminoacyloxy, carboxyacyloxy, alkoxycarbonyloxy, haloacyloxy, amino and monoand disubstituted amino, useful as antibacterial agents and in promoting growth and improving feed efficiency of animals in general.

United States Patent [191 Cronin et al.

[ Sept. 23, 1975 SUBSTITUTED ALKYL ESTERS QUlNOXALINE-DI-N-OXIDE-2- CARBOXYLIC ACID AS GROWTH PROMOTING AGENTS [75] Inventors: Timothy H. Cronin, East Lyme;

Kenneth Richardson, Groton, both of Conn.

[73] Assignee: Pfizer, Inc., New York, NY.

[22] Filed: Sept. 13, 1973 [21] Appl. No.: 397,163

Related US. Application Data [60] Division of Ser. No. 135,792, April 20,1971, Pat. No. 3,818,007, which is a continuation-in-part of Ser. No. 20,841, March 18, 1970, abandoned.

[52] US. Cl. 424/250; 260/250 QN [51] Int. Cl. A61K 31/495 [58] Field of Search 424/250; 260/250 [56] References Cited UNITED STATES PATENTS 3,344,022 9/1967 Johnston 424/94 8/1972 Song et al. 424/250 8/1973 Abushanab 424/250 OTHER PUBLICATIONS Chemical Abstracts, Vol. 75 (1971), p. 98591q.

Primary Examiner-V. D. .Turner Attorney, Agent, or Firm-Connolly and Hutz [57] ABSTRACT Novel alkyl esters of quinoxaline-di-N-oxide-2- 'carboxylic acid substituted on the alkyl portion of the 1 Claim, No Drawings SUBSTITUTED ALKYL ESTERS QUINOXALINE-DI-N-OXIDE-2-CARBOXYLIC ACID AS GROWTH PROMOTING AGENTS CROSS REFERENCE TO RELATED APPLICATIONS BACCKGROUND OF THE INVENTION This invention relates to quinoxaline-di-N-oxides, and more particularly to a unique series of 3- methylquinoxaline-di-N-oxide-2-carboxylic acid alkyl esters, substituted on the alkyl portion of the ester by hydroxy, acyloxy, N-alkyl carbamyloxy, dialkylaminoacyloxy, carboXyacyloxy, alkoxycarbonyloxy, haloacyloxy, amino and'monoand disubstituted amino and the appropriate basic, quaternary and acid addition salts thereof possessing antibacterial activity against pathogenic microorganisms and to methods for the promotion of weight gain and food consumption of animals.

Continuing efforts to uncover new and more useful antibacterial agents have led, over the years, to the development of a wide variety of prototype organic compounds including numerous congeners'of quinoxalinedi-N-oxides. Landquist, et al., J. Chem. Soc., 2052 (1956), in a search for compounds of improved antibacterial or antiprotoz oal activity, reported the preparation of several derivatives of 2-methy1- and 2,3- dimethylquinoxaline di-N-oxides in which the methyl groups were converted to groups such as bromomethyl acetoxymethyland hydroxymethyl 7 including 3- methyl-Z-carbethoxyquinoxaline-di-N oxide. However, no utility is alleged for any of these compounds. French Pat. No; M3717, granted Jan.'3,-19'66,- dis closes 2,-.

quinoxalinecarb'oxamide-di-N oxides in' which the carboxamide group may be substituted with an alkyl, substituted alkyl, aryl, aralkyl, or cycloalkyl group. Also disclosed, but the structure not indicated, are the corresponding 2-quinoxalinecarboxylic acid substituted esters. They are reported to be of use in human therapy as antitubercular, antibacterial, anticancer, antivirus and antiprotozoal agents.

Belgian Pat. No. 697,976, granted Nov. 3, 1967, describes a variety of N substituted derivatives of 3- methyl-Z-quinoxalinecarboxamide-di-Naoxide in which the N-substituent is-phen'yl, substituted phenyl, dodecyl or ethyl, as well as the corresponding 3-i'nethyl-2- carbethoxyquinoxaline-di-N-oxide. They are said to be of value as intermediates for the preparation of vegetation protection agents and-pharmaceutical agents. Belgian Patent Nos. 721,724; 721,725; 721,726; 721,727 and 721,728; published Apr. 2, 1969, describe a variety of N-substituted 3-methy1-2-quinoxalinecarboxamide-di-N-oxide derivatives wherein the N-substituent is a hydroxyalkyl, lower alkoxyalkyl, carboalkoxyalkyl,

monoalkylaminoalkyl or di(alkyl)amirioalkyl group as antibacterial agents. -a- 1 I SUMMARY OF THE iNvENnoN .The novel antibacterial and' growth promotant quinoxaline-di-N-oxides of thisinvention are represented by the formula: i

wherein X is a substituent at the 6- or 7-position selected from the group consisting of hydrogen, fluorine,

. chlorine, bromine, trifluoromethyl, methyl and methoxy;

A is alkylene of from 2 to 5 carbon atoms; and

Z is selected from the group consisting of: (l) hydroxy; (2) acyloxy of the formula -O CR wherein R is selected from the group consisting of hydrogen; alkyl containing from 1 to 10 carbon atoms; 1

alkoxy containing from 1 to 4carbon atoms; alkylamino containing from 1 to 4 carbon atoms; car boxyalkyl containing from 2 to 8 carbon atoms;

and substituted alkyl wherein said alkyl contains.

from l to' 3 carbon atoms and said substituent is seacid addition salts thereof wherein Z is selected from the group consisting of amino, mono substituted amino, disubstituted amino, and ylarninoacyloxy; pharmaceutically acceptable basicsalts thereof wherein Z is carboxyacyloxy; and loweralkyl pharmaceutically acceptable qua ternary, salts thereof wherein Z is dialkylaminoacyloxy. v

3 Of particular interest, because of their in vitro and in 'vivo antibacterial activity and animal growth promotant activity, are compounds wherein X is hydrogen or chlorine, A is alkylene of from 2 to 3 carbon atoms and Z is hydroxy, acetoxy, n-octanoyloxy, (ethoxycarbonyl- )oxy, 3-carboxypropionyloxy, 4-carboxybutyryloxy, amino, dimethylamino -or methylethylamino.

Also considered within the scope of this invention are congeners of the aforementioned compounds where the 3-'rnethyl' substituent is replaced by lower alkyl containing from 2 to 3 carbon atoms; where Z is acyloxy of the formula O CR, wherein R is phenyl and substituted phenyl, the substituent being selected from the group consisting of halo (F, Cl, Br), lower alkoxy,

lower alkyl and lower dialkylamino; where A is part of a cycloalkyl group containing from 3 to 8 carbon atoms and where A, as alkylene or part of a cycloalkyl group is substituted by hydroxy. Also included within the purview of thenpresent application are those congeners wherein the acyl portion of. the 'carboxyacyloxy group, Z, is derived from a cycloalkylcarboxylic acid or an aromatic acid, including benzoic acids, napthalenecarb'oxylic acids and heterocyclic acids. Also contemplated is polycarboxyacyloxy as being represented by the variable Z. in like manner these compounds also possess antibacterial and growth promotant activity.

lected from the group consisting of chlorine, bro- 1 dialk- 3 4 DETAILED DESCRIPTION OF THE INVENTION Quinoxalinedi-N-oxides of the instant invention re- In accordance with the process employed for preparsult from the condensation of benzofuroxan and substiing 3-methyl-2-quinoxalinecarboxylic acid alkyl esters tuted benzofuroxans with acetoacetic esters such that of the instant invention, an appropriately substituted the 2- and 3-positions of the resulting annellated strucbenzofuroxan is contacted with an acetoacetic ester de- 5 ture represent the carbonyl carbon and the carbon of rivative as illustrated in the following scheme: the active methylene group of the acetoacetate.

O l N cn CO AZ s x O C=O x I N CH, I

o O 0 A Z wherein X and A are as previously defined, and Z is al- The substituents on the benzene moiety of the 3- kanoyloxy or disubstituted amino. methyl-2-quinoxalinecarboxylic acid ester-l,4-dioxides As a necessary element of the herein described procan vary widely. For example, at least one of the folcess, the reaction sequences described above must be lowing substituents can be present: hydrogen, methyl, effected in the presence of a base. Such a base is of varmethoxy, chloro, fluoro, bromo and trifluoromethyl. In ied character. For instance, it is meant to contemplate like manner, the substituents may include methylthio,

such bases as organic amines, ammonia, alkali metal methylsulfonyl, methylsulfinyl, trifluoromethylthio, trihydroxides, hydrides and alkoxides. Representative of fluoromethoxy, acetyl, amino, nitro, dimethylamino,

such bases are ammonia, primary amines such as nacetamido, sulfamyl and monoand dimethylsulfamyl, propylamine, n-butylamine, aniline, cyclohexylamine, methylsulfonylamino, mercapto, hydroxy, acetoxy, carbenzylamine, p-toluidine, ethylamine, octylamine, terboxy, carboxamido and monoand dimethylcarboxtiary amines such as diethylaniline, N- amido, cyano, aldehydo and phosphone. The favored methylpyrrolidine, N-methylmorpholine, and 1,5- positions on the fused benzene ring of said final proddiazabicyclo[4,3,0]-5-nonene; sodium hydroxide, ponets are the 6- or 7-positions. The favored positions for tassium hydroxide, ammonium hydroxide, sodium substituents on the aryl ring of the starting benzofuroxethoxide, potassium methoxide, and sodium hydride. ans leading to said final products are the 5- or 6- The preferred base to use is an organic amine, ammopositions. When one of said substituted benzofuroxans niaor alkoxide. is condensed with the requisiteacetoacetate, a 6- and In practice, a solution or suspension of the appropri- 7-substituted quinoxaline-di-N-oxide are produced. ately substituted benzofuroxan and the requisite aceto- This multiple product formation results because of the acetate in a reaction-inert solvent such as ethano two orientation possibilities of the acetoacetate frag- N,N-dimethylformamide, benzene, tetrahydrofuran, ment in the final product For example, if one reacts a chloroform or hexamethylphosphoramide is treated 5-substituted benzofuroxan of the formula;

with an alkoxide, e.g'., sodium ethoxide. It is preferable to use at least an equimolar amount of the benzofuroxan and acetoacetate, while the amount of base X N may be from a catalytic to equimolar amount. The re- U action is carried out at ambient temperatures, although it may be heated to 100C. to hasten product forma- 0 tion. Reaction time is not critical, but will vary depending on the reactivity of the starting materials, temperature and solvent employed. Substantial yields of the deith a reactant CH COCH CO -AZ, two products, sired products are isolated with reaction periods of 15 a 6- d 7- b i d i li -di-N- id result 35 minutes to 24 hours. shown by the formulae:

3 i x CO2-AZ x N\ CH3 and 2 CO2A-Z O I The requisite benzofuroxans and acetoacetates are If the corresponding 6- substituted benzofuroxan is either readily available or easily prepared by those employed as the starting material, the same two possiskilled in the art. For instance, the synthesis of varible products are formed.

ously substituted benzofuroxans is described by Kauf- The mixture of isomers is recovered by methods man, et al., in Advan. Heterocyclic Chem., 10, l (1969). known to those skilled in the art. In many of the prepa- Acetoacetates are readily prepared from diketene acrations disclosed wherein a solid, often crystalline macording to the general procedure of Brintzinger, et al., terial, separates from the reaction mixture, the solid ap- Chem. Ber., 83, 103 (I950). pears to consist predominantly of one of the isomers.

Said isomer can be purified by repeated recrystalliamino, -NH are synthesized by the following scheme:

zation from a suitable solvent to a constant melting point. The other isomer, the one present in smaller amounts in the originally isolated solid material, is the predominant product in the mother liquor. It can be recovered therefrom by methods known to those skilled in the art, as for example, the evaporation of the mother liquor and repeated crystallization of the residue to a product of constant melting point. Alternatively, the reaction mixture can be extracted either before or after evaporation to dryness.

The identification of the isomers has not been completed. Both isomers of a given compound, however, exhibit the same type of activity, e.g., as animal growth promotants or as antibacterial agents.

Compounds of the present invention wherein Z is hydroxy are most conveniently prepared from the requisite acyloxy analogs by acid hydrolysis. in practice, a 3-methyl-2-quinoxalinecarboxylic acid alkyl ester, 1,4- dioxide substituted in the alkyl portion of the ester by an alkanoyloxy moiety is added to an aqueous acid solution, for instance, sulfuric, phosphoric, or hydrochloric. In general, the concentration of the acid is from about 1-12N, with a preferred range of 10-12N. The hydrolysis is effected at temperatures of from 050C. with a preferred temperature of 2535C. and a reaction period of 30 minutes to 3 hours. After the reaction is completed water is added to the mixture and the pH is adjusted to pH 5 using an aqueous solution of a suitable base, e.g., sodium hydroxide. The mixture is then extracted several times with a suitable solvent such as chloroform, and the organic layer separated, dried over sodium sulfate and conentrated in vacuo to dryness.

Compounds of the present invention wherein Z is Hydrolysis of the aforementioned oxazacyclic moiety at the 2-position of the appropriate 3- methylquinoxaline-l ,4-di-N-oxide is most conveniently carried out using a suitable acid, such as hydrobromic, hydrochloric, phosphoric or sulfuric in a water-water miscible solvent system such as methanol-water or ethanol-water. In general, a two to five fold excess of said acid is employed to facilitate the reaction. The hydrolysis is carried out at temperatures of 050C. with a preferred range of 25 35C. and for a reaction period of 15 minutes to 2 hours.

The workup procedure for said reaction consists of removal of excess water, solvent and acid under reduced pressure, followed by trituration of the resulting salt with a suitable solvent, such as ethyl acetate or isopropanol. It is advantageous, whenever possible, to employ the same acid for the hydrolysis as is desired as the salt of the final product. For example, if the hydrochloride salt is desired then hydrochloric acid is employed, the sulfate salt-sulfuric acid, etc.

The requisite 2-( l,3-oxazacyclic)-3- methylquinoxaline-l ,4-di-N-oxides for the aforementioned hydrolysis reaction are conveniently prepared by one of two synthetic routes. The first scheme, as illustrated, consistsof the with an appropriate solvent, such as chloroform or methylene chloride.

The N-substituted acetoacetamide derivatives employed, as starting materials are easily synthesized by those skilled in the art as taught by the method of DAngeli, et al., Tetrahedron Letters, 605 (1965 The second and alternate synthesis of the intermediate 2-( l ,3-oxazacyclic)-3-methylquinoxaline-1 ,4-di-N- oxides comprises contacting an appropriately substi- The aforementioned N-ttuted benzofuroxan with a 2-acetonyl-l,3-0xazacyclic butyloxycarbonylaminoalkanols are easily prepared compound, as illustrated: from the aminoalkanols and t-butyl cyanoformate by 1 O N N n N X CH CCH A x A s o a L CH. O i o The reaction conditions, solvents and isolation leadprocedures known in the art; for example, by the gening to these compounds are the same as previously outeral procedure of Leplawy, et al., Bull. acad. polon. sci., lined for the products of the present invention. ser. sci. chem., 12, 21 (1964); C. A., 61, 1933 (1964). Acylation of the anion of the appropriate 2-methyl- The second step in the sequence which relates to the 1,3-oxazacyclic intermediate employing either ethyl removal of the t-butyl protecting group is carried out acetate or acetyl chloride provides the desired 2- using a dilute mineral acid such as hydrochloric acid or acetonyl derivative. In practice, the anion of the requihydrobromic acid. In practice, the 3-methyl-2- site Z-methyl-l ,3-oxazacyclic compound generated acquinoxalinecarboxylic acid, N-tcording to the conditions as taught by Meyer, et al., J. butyloxycarbonylaminoalkylene ester, 1,4-dioxide in a Am. Chem. Soc., 91, 763 (1969), is treated with an suitable, water miscible solvent such as ethanol or equimolar amount of ethyl acetate or acetyl chloride in methanol is treated with at least one mole equivalent of a suitable solvent such as tetrahydrofuran or diethyl said acid and preferably a 10-50% excess. The reaction ether at temperatures of from 30 to 50C. When the is carried out in a temperature range of 050C with reaction is complete, it is filtered and the solvent rea Preferred reaction temperature of and moved in vacuo. The desired acetonyl derivative i pu-g 25 reaction period of minutes to 3 hours. A convenient rified either by distillation under r du ed pressure or method of isolation consists of removal of the solvent recrystallized from a suitable solvent. and excess acid under reduced pressure. The resulting The aforementioned 2-methyl-l ,3-oxazacyclic comsalt can be further purified by recrystallization from or pounds necessary for the aforedescribed procedure are trituration with a suitable solvent. either readily available or ea ily r d by method 30 In addition to the aforementioned synthetic routes to known to those skilled in the art, for instance, accordthe compounds of the present invention wherein Z is ing to the methods as outlined by Eld fi ld, Heteracyloxy and disubstituted amino, there are three addicyclic Compounds, John Wiley and Sons, Inc., New tional preparative schemes. York, 1957, Vol, 5, page 377 and Vol, 6, ge 534, and Scheme I encompasses the condensation of a benb E k i Ad yeterocyclic Chem 311 1963) zofuroxan with an a-ketoester, illustrated as follows:

0 0 l l u @fKy X o R CH,CCO A-Z x I N N 1 1 0 Compounds of the instant invention where Z is a sec-- wherein X and A are as previously indicated, R is alkyl ondary amine, NllR wherein R is as previously in- 5 containing from 1 to 3 carbon atoms and Z is acyloxy dicated, are prepared by the following sequence of reof the formula O CR where R is hydrogen, alkyl conactions: taining from 1 to 10 carbon atoms, or benzoyl and sub- N CH 1 l a N I O CO A-NCO :)a -{jli I (I: R 7 CH3 0 o \O--ANCO2C(CH;,)3 o O H* I CH,=C -CH C02A-NHR,

I X -cof I CH3 Reaction conditions, solvents and isolation procestituted benzoyl; Z is disubstituted amino. dures of the first reaction relating to the formation of The reaction conditions and solvents for Scheme 1 the quinoxaline-di-N-oxide from the requisite benare essentially those described previously for the conzofuroxan and acetoacetate are as previously dedensation of a benzofuroxan and an acetoacetate ester, scribed, as are the reaction conditions leading to the with the preferred base an alkali metal alkoxide. The acetoacetates from diketene and the appropriate N-tworkup procedure and product isolation are as previbutyloxycarbonylaminoalkanol. ously indicated.

wherein X is as previously indicated and M is lower alkyl or phenyl.

In practice, the starting lower alkyl or phenyl ester is treated with at least an equimolar, and preferably a IOU-200% excess of the requisite alkanol, HO-A-Z, and an acid, e.g., toluenesulfonic acid or hydrochloric acid. Said acid may be used in catalytic quantities or in as much as an equimolar amount plus a -20% excess. The reaction may be carried out either in a solvent such as benzene, toluene or xylene or neat, i.e., without a solvent. However, it is preferred that a solvent be used to provide a single contact phase. The reaction temperature used will vary with the reflux temperature of the solvent employed. In general, temperatures of 90l 10C. are adequate to effect the aforementioned reaction, with reaction times dependent on the dilution of the reaction mixture and the reactivities of the starting reagents. The products are isolated by removing the solvent in vacuo, followed by trituration or recrystallization from an appropriate solvent." The requisite 3- aIkyl-Z-quinoxalinecarboxylic acid esters used as a starting reagent in Scheme II are prepared according to the method of Issidorides, et al., J. Org. Chem., 31, 4067 (1966). I

The third alternate preparative method, Scheme III, employs the alcoholysis of the corresponding 2- cyanoquinoxaline-di-N-oxides as follows:

0 l CN fiII &.

wherein X, R A and Z are as previously indicated.

In practice, the nitrile is heated with at least an equimolar amount and as much as al00% excess of the requisite alkanol in the presence of at least anequimolar amount plus as much as a excess of an acid such as sulfuric or hydrochloric acid 'ina reaction-inert solvent or neat. A necessary element in the described reaction is at least an equimolar amount of water. In general, steam bath temperatures are adequate to effect said reaction. Reaction times are not critical, but will vary with temperature and the reactivity of the starting reagents.

The work-up procedure employs removal of excess alkanol in vacuo followed by trituration of the product with a suitable solvent.

The requisite 2-cyano-3-alkylquinoxaline-di-N- oxides are synthesized from the corresponding benzofuroxan and the appropriate B-ketonitrile. Said B-ketonitriles are readily ascessable' by methods known to those skilled in the art, e.g., according to the procedures as outlined in Synthetic Organic Chemistry,- John Wiley & Sons, Inc., New York, 1956, page 348.

Compounds of the instant invention wherein Z is carboxyacyloxy and X and A are as previously indicated are synthesized from those wherein Z is hydroxy via acylation of said hydroxy moiety employing an activated form of a dicarboxylic acid. Said activation may be through the acid halide, mixed anhydride or the simple cyclic 'anhydride, all in the presence of a proton accep- 'tor such as a tertiary amine, i.e., triethyl amine. Also useful in the aforedescribed condensation utilizing the dicarboxylic acid and alcohol is the condensing agent carbodiimide.

In practice, the hydroxyalkyl ester of 3-methyl-2- quinoxalinecarboxylic acid I,4-dioxide is acylated with at least an equimolar amount, plus as much as a excess of an activated dicarboxylic acid selected from the group previously enumerated. Said reaction is conducted in an aprotic solvent such as acetone, methylene chloride or chloroform. Reflux temperatures are favored, with reaction times of 2-6 hours. As previously mentioned, a proton acceptor is employed in molar amounts equivalent to the dicarboxylic acid.

The reaction is subsequently quenched in water, extracted with a water immiscible solvent and the water layer acidified with concentrated hydrochloric acid to a pH of about 3. The liberated product is extracted into chloroform and the organic layer separated, dried over sodium sulfate and concentrated in vacuo to dryness. The residual product can be further purified by recrystallization from an appropriate solvent.

Acylation of the hydroxy] moiety with alkyl chloroformate esters leads to those analogs wherein Z is alkoxycarbonyloxy, and is carried out employing the appropriate hydroxyalkyl ester and a halo formate ester in a solvent such as chloroform or methylene chloride in the presence of a tertiary amine, e.g., pyridine or triethyl amine. Generally, it is advantageous to employ as much as a 100% excess of the halo formate and tertiary amine. I

Experimentally, a solution of the hydroxyalkyl ester and tertiary amine,.cooled to 0C. is treated with the requisite halo formate. After l-2 hours at ambient temperatures the reaction mixture is treated with a saturated sodium bicarbonate solution then washed with 6N hydrochloric acid. The solvent, containing the product,- is then dried and evaporated to dryness. The product is usually triturated or recrystallized from a suitable solvent.

In a similar manner, congeners of the products of the present invention wherein Z is chloroor bromoacyloxy and N-alkylcarbamyloxy are synthesized by contacting the requisite hydroxyalkyl ester with a chloroor bromoacyl' halide or alkylisocyanate, respectively.

Reaction of the aforementioned chloroor bromoacyloxy compounds with at least two moles of a secondary amine in a reaction-inert solvent such as benzene, chloroform or methylene chloride at 50-80C. for 1-3 hours results in the preparation of those congeners wherein Z is dialkylaminoacyloxy.

These resulting tertiary amines are readily quaternarized by treatment with 'at least an equimolar amount of an appropriate alkylating agent, e.g., alkyl halide or alkyl sulfonate ester. Said reaction is most conveniently carried out at elevated temperatures in a reaction-inert solvent such as acetone, tetrahydrofuran or benzene. The product, which generally forms as a precipitate as the reaction proceeds, is filtered from the cooled reaction mixture and subsequently recrystallized.

An alternate, and equally effective means of preparing these quaternary salts, comprises the substitution of a tertiary amine for a secondary amine in the aforementioned reaction with compounds wherein Z is chloroand bromoacyloxy. In said reaction, compounds of the present invention wherein Z is chloroand bromoacyloxy dissolved in methylene chloride or chloroform are treated with at least a molar equivalent, plus as much as a 100% excess, of the appropriate trialkylamine at ice-bath temperatures for from 6-12 hours. The resulting quaternary chloride or bromide salts are conveniently isolated by filtration from the reaction mixture.

As has been previously noted, the compounds of the instant invention wherein Z is amino, monoand disubstituted amino and dialkylaminoacyloxy can form acid addition salts wherein said acid addition salts are considered to be the full equivalent of the free bases. Basic compounds of the present invention are converted to the acid addition salts by interaction of the base with an acid either in an aqueous or non-aqueous medium. In a similar manner, treatment of the acid addition salts with an aqueous base solution, e.g.. alkali metal hydroxides, alkali metal carbonates and alkali metal bicarbonates or with a metal cation which forms an insoluble precipitate with the acid anion, results in a regeneration of the free base form. Such conversions are best carried out as rapidly as possible and under temperature conditions and method dictated by the stability of said basic products. The bases thus regenerated may be reconverted to the same or a different acid addition salt.

In the utilization of the chemotherapeutic activity of those compounds of the present invention which form salts, it is preferred, of course, to use pharmaceutically acceptable salts. Although water-insolubility, high toxicity, or lack of crystalline nature may make some particular salt species unsuitable or less desirable for use as such in a given pharmaceutical application, the water insoluble or toxic salts can be converted to the corresponding pharmaceutically acceptable bases by decomposition of the salt as described above, or alternately they can be converted to any desired pharmaceutically acceptable acid-addition salt.

Examples of acids which provide pharmaceutically acceptable anions are hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric, or sulfurous, phosphoric, acetic, lactic, citric, tartaric, succinic, maleic and gluconic.

A characteristic feature of the compounds of the present invention wherein Z is carboxyacyloxy and A and X are as previously indicated is their ability to form basic salts. Said compounds are converted to basic salts by the interaction of said acid with an appropriate base in an aqueous or non-aqueous medium. Such basic reagents suitably employed in the preparation of said salts can vary in nature, and are meant to contemplate such bases as organic amines, ammonia, alkali metal hydroxides. carbonates, bicarbonates, hydrides and alkoxides, as well as alkali earth metal hydroxides, hydrides, alkoxides and carbonates. Representative of such bases are ammonia, primary amines such as n-propylamine, n-butylamine, aniline, cyclohexylamine, benzylamine, p-toluidine, ethylamine, octylamine, tertiary amines such as diethylaniline, N-methylpyrrolidine, N- methylmorpholine and I,5-diazabicyclo-[4,3,01-5- nonene; sodium hydroxide, potassium hydroxide, ammonium hydroxide, sodium ethoxide, potassium methoxide, magnesium hydroxide, calcium hydride and barium hydroxide.

As previously indicated, the quinoxaline-di-N-oxides of the present invention are all readily adapted to therapeutic use as antibacterial agents and as growth promotants. Typical member compounds of interest in this series include 3-methyl-Z-quinoxalinecarboxylic acid, 2-aminoethyl ester, l,4-dioxide; 3-methyl-2- quinoxalinecarboxylic acid, 2-(dimethylamino)ethyl ester, l,4-dioxide; 3-methyl-2-quinoxalinecarboxylic acid, 2-(methylethylamino)ethyl ester, l,4-dioxide, 3--

methyl-2-quinoxalinecarboxylic acid, 2- (acetyloxy)ethyl ester, l,4-dioxide; 3-methyl-2- quinoxalinecarboxylic acid, 2-hydroxyethyl ester, l,4- dioxide; 3-methyl-2-quinoxalinecarboxylic acid, 2-(3- carboxypropionyloxy)ethyl ester, l,4-dioxide; 3- methyl-2-quinoxalinecarboxylic acid, 2-(4-carboxybutyryloxy)ethyl ester, l,4-dioxide; 3-methyl-2- quinoxalinecarboxylic acid, 2-(octanoyloxy)ethyl ester, 1,4-dioxide; 3-methyl-2-quinoxalinecarboxylic acid, 2-[(ethoxycarbonyl)oxy]ethyl ester, l,4-dioxide; 3-methyl-2'quinoxalinecarboxylic acid, 3- (acetyloxy)propyl ester, l,4-dioxide, 3-methyl-2- quinoxalinecarboxylic acid, 3-hydroxypropyl ester, 1,4-dioxide; 3-methyl-2-quinoxalinecarboxylic acid, l-methyl-Z-hydroxyethyl ester, l,4-dioxide; 3-methyl- 2-quinoxalinecarboxylic acid, 3- (dimethylamino)propyl ester, l,4-dioxide; 3-methyl-6- and 7-chloro-2-quinoxalinecarboxylic acid, l-methyl- Z-(diethylamino )ethyl ester, l,4-dioxide, and 3-methyl- 6- and 7-chloro-Z-quinoxalinecarboxylic acid, 2- (acetyloxy)ethyl ester, l,4-dioxide.

The valuable products of this invention are readily effective in treating a wide variety of pathogenic microorganisms. They are, therefore, useful as industrial antimicrobials, for example, in water treatment, slimecontrol, paint preservation and wood preservation as well as for total application purposes as disinfectants.

For in vitro use, e.g., for topical application, it will often be convenient to compound the selected product with a pharmaceutically acceptable carrier such as vegetable or mineral oil or an emollient cream. Similarly, they may be dissolved or dispersed in liquid carriers or solvents such as water, alcohol, glycols or mixtures thereof or other pharmaceutically acceptable inert media, that is, media which have no harmful effect on the active ingredient. For such purposes, it will generally be acceptable to employ concentrations-of active ingredients of from about 0.01 percent to about 10 percent by weight based on total composition.

In determining the in vitro activity of an antibiotic, the sensitivity of the various micro-organisms to an antibiotic is determined by the commonly accepted twofold serial dilution technique. Final concentrations of compound per ml. range from 100 mcg. in the first tube to 0.19 mcg. in the tenth tube. The inoculum consists of 0.5 ml. of a l X 10 dilution of a standardized culture. Final volume in each tube or cup in the DisPoso tray is 1.0 ml. The tubes are incubated at 37C. for approximately 24 hours. The medium used is Witkins synthetic or Brain Heart Infusion (BI-II). The sensitivity (MIC minimal inhibitory concentration) of the test organism is accepted as evidenced by the absence of gross turbidity.

Further, compounds described herein exhibit useful broad spectrum activity, that is, activity against both gram-negative and gram-positive bacteria, in contrast to the usual gram-negative activity of quinoxaline-di-N- oxides. Additionally, the compounds of the present invention are active in vivo and are especially useful as animal growth promotants, especially for swine and poultry.

When used in vivo for such purposes, these novel compounds can be administered orally or parenterally, e.g., by subcutaneous, intramuscular or intravenous injection, at a dosage of from about 1 mg./kg. to about 100 mg./kg. of body weight. Vehicles suitable for parenteral injection may be either aqueous such as water, isotonic saline, isotonic dextrose, Ringers solution, or non-aqueous such as fatty oils of vegetable origin (cotton seed, peanut oil, corn, sesame), dimethylsulfoxide and other non-aqueous vehicles which will not interfere with therapeutic efficiency of the preparation and are nontoxic in the volume or proportion used (glycerol, propylene glycol, sorbitol and dimethylacetamide). Additionally, compositions suitable for extemporaneous preparation of solutions prior to administration may advantageously be made. Such compositions may include liquid diluents, for example, propylene glycol, diethyl carbonate, glycerol, sorbitol, etc.; buffering agents, hyaluronidase, local anesthetics and inorganic salts to afford desirable pharmacological properties. These compounds may also be combined with various pharmaceutically acceptable inert carriers including solid diluents, aqueous vehicles, nontoxic organic solvents in the form of capsules, tablets, lozenges, troches, dry mixes, suspensions, solutions, elixirs and parenteral solutions or suspensions. In general, the compounds are used in various dosage forms at concentration levels ranging from about 0.5 percent to about 90 percent by weight of the total composition.

The in vivo efficacy of the compounds of the instant invention is determined by the antibacterial activity against acute infections in mice. The acute experimental infections are produced by the intraperitoneal inoculation of standardized culture suspended in either hog gastric mucin or broth. A brief discussion of the words standardized culture would seem to be beneficial. In order to obtain reproducible results with a test compound it is necessary to control, as much as possible, the many variables that can enter into this type of test. An organism of high virulence if used in large enough numbers can make almost any drug look inactive. On the other hand, an inoculum not able to produce a measurable difference between treated and untreated groups is equally lacking in purpose.

Stock cultures of test organisms are normally maintained on slants or in liquid medium. When not routinely used they are maintained at refrigerator temperature or in a lyophilized state. When it becomes necessary to use a culture in animal protection tests the culture is suspended in a volume of saline or broth, and the density of the suspension is measured by a photoelectric colorimeter. From this stock, lO-fold dilutions are prepared. Each dilution is inoculated into a series of mice in order to determine the LD the LD being the lowest concentration of organisms required to produce 100 percent deaths. For example, if it is found that a dilution of 10 is the lowest level of organism that will produce 100 percent death, we would probably use an inoculum of 10 for the drug evaluation experiments. This means that we are using about 10 LD or 10 times the minimum dose required to kill mice. Such a test would also include the use of control animals whwich receive an inoculum of 10" 10' and possibly 10*. These dilutions are included as a check on possible variation in virulence which can occur. Having previously determined, through the virulence titration, that 10 was the maximum dilution that will kill we naturally expect these animals to die, usually within 24 hours.

Each organism has its own standardized inoculum level. Some, such as Staphylococcus, may be used at 10, while others like Streptococcus require weekly animal passage in order to maintain virulence.

When evaluating an antibiotic for its effectiveness after a single dose, the dose is usually administered 0.5 hour after inoculating the mice with the lethal concentration of organisms. In this type of treatment schedule surviving mice are usually held for four days after the treatment and the percent alive is calculated.

Other methods of administration of the useful products of this invention to animals include mixing with animal feeds, the preparation of feed concentrates and supplements and dilute solutions or suspensions, e.g., a 0.1 percent solution, for drinking purposes. Surprisingly, the addition of low level amounts of the herein described quinoxaline-di-N-oxides to the diet of healthy animals, both ruminant and non-ruminant, such that these animals receive the product over an extended period of time, at a level of from about 0.1 mg./kg. to about 100 mg./kg. of body weight per day, especially over a major portion of their active growth period, results in-an acceleration of the rate of growth and improves feed efficiency (the number of pounds of feed required to produce a pound gain in weight). Included in these ;two classes of animals are poultry (chickens, ducks, turkeys), cattle, sheep, dogs, cats, swine, rats, mice, horses, goats, mules, rabbits, mink, etc. The beneficial effects in growth rate and feed efficiency are over and above what is normally obtained with complete nutritious diets containing all the nutrients, vitamins, minerals, and other factors known to be required for the maximum healthy growth of such animals. The animals thus attain market size sooner and on less feed.

The herein described feed compositions have been found to be particularly valuable and outstanding in the case of swine. In some insances the degree of response may vary with respect to the sex of the animals. The

products, may, of course, be administered in one component of the feed or they may be blended uniformly throughout a mixed feed; alternatively as noted above, they may be administered in an equivalent amount via the animal s water ration. It should be noted that a variety of feed components may be of use in the nutritionally balanced feeds. Any animal feed composition may be prepared to comprise the usual nutritional balance of energy, proteins, minerals and vitamins together with one or more of the quinoxaline-di-N-oxides described above. Some of the various components are commonly grains such as ground grain and grain byproducts; animal protein substances, such as meat and fish by-products, vitaminaceous mixtures, e.g., vitamin A and D mixtures, riboflavin supplements and other vitamin B complexes; and bone meal, limestone and other inorganic compounds to provide minerals.

The relative proportions of the present compounds in feeds and feed concentrates may vary somewhat, depending upon the compound, the feed with which they are employed and the animal consuming the same. These substances are advantageously combined in such relative proportions with edible carriers as to provide pre-mixes or concentrates which may readily be blended with standard nutritionally balanced feeds or which may be used themselves as an adjunct to normal feedings.

In the preparation of concentrates a wide variety of carriers, including the following: soybean oil meal, corn gluten meal, cotton seed oil meal, sunflow seed meal, linseed oil meal, cornmeal, limestone and corncob meal can be employed. The carrier facilitates uniform distribution of the active materials in the finished feed with which the concentrate is blended. The concentrate may be surface coated, if desired, with various proteinaceous materials, or edible waxes, such as zein, gelatin, microcrystalline wax and the like to provide a protective film which seals in the active ingredients. It will be appreciated that the proportions of the drug preparation in such concentrates are capable of wide variation since the amount of active materials in the finished feed may be adjusted by blending the appropriate proportion of concentrate with the feed to obtain the desired degree of supplementation. In the preparation of high potency concentrates, i.e., pre-mixes, suitable for blending by feed manufacturers to produce finished feeds or concentrates of lower potency, the drug content may range from about 0.1 g. to 50 g. per pound of concentrate. The high potency concentrates may be blended by the feed manufacturer with proteinaceous carriers, such as soybean oil meal, to produce concentrated supplements which are suitable for direct feed ing to animals. The proportion of the drug in these supplements may vary from about 0.1 to 10 g. per pound of supplement. A particularly useful concentrate is provided by blending 2 g. of drug with 1 pound of limestone or 1 pound of limestone-soybean oil meal (1:1). Other dietary supplements, such as vitamins, minerals, etc., may be added to the concentrates in the appropriate circumstances.

The concentrates described may also be added to animal feeds to produce a nutritionally balanced, finished feed containing from about 5 to about 125 g. of the herein described compounds per ton of finished feed. In the case of ruminants, the finished feed should contain protein, fat, fiber, carbohydrate, vitamins and minerals, each in an amount sufficient to meet the nutritional requirements of the animal for which the feed is intended. Most of these substances are present in naturally occurring feed materials, such as alfalfa hay or meal, cracked corn, whole oats, soybean oil meal, corn silage, ground corn cobs, wheat bran and dried molasses. Bone meal, limestone, iodized salt and trace minerals are frequently added to supply the necessary minerals and urea to provide additional nitrogen.

, mix). The pigs are divided into groups of 32 pigs each As is well known to those skilled in the art, the types of diets are extremely variable, depending upon the purpose, type of feeding operation, species, etc. Specific diets for various purposes are listed by Morrison in the Appendix of Feeds and Feeding, the Morrison Publishing Company, Clinton, Iowa, 1959.

In the case of non-ruminant animals, such as hogs, a suitable feed may contain from about 50 to percent of grains, 3 to 10 percent animal protein, 5 to 30 percent vegetable protein, 2 to 4 percent of minerals, together with supplementary vitaminaceous sources.

In practice, growth promotion is determined in swine, for instance, by the method wherein young pigs from 5-6 weeks of age and averaging 21.5 lbs. initial body weight are maintained on an ad libitum consumption of water and feed formulation consisting of ground yellow corn (58.1%), soybean meal (19.6%), alfalfa meal (2.0%), dried skim milk(5.0%), dried whey (10.0%), stabilized animal fat (2.5%), limestone (0.6%), dicalcium phosphate (1.1%), iodized salt (0.5%), vitamin premix PPM No. 5 (0.5%), quadruple delamix (0.05%), and zinc carbonate (156 g./2000 lbs.

and are held for a pre-experimental period of three days prior to starting the experiment. The quinoxalinedi-N-oxides of the present invention are added to the feed at a ratio of 50 g. of compound per ton of feed. After 28 days the efficacy of said added compounds on growth promotion is measured by a comparison of the growth in terms of weight gain of the treated animals with the untreated control group, which is arbitrarily assigned a weight gain Index of 100. For example, if a compound effects a growth 23% greater than the control group (with an Index of it would be assigned a value of 123, etc.

The effect of a supplemented diet on the growth promotion in other species of animals using appropriate dose levels and feed formulations is assessed in an analogous manner.

The following examples are provided solely for the purpose of illustration and are not to be construed as limitations of this invention, many variations of which are possible without departing from the spirit or scope thereof.

EXAMPLE 1 3-Methyl-2-quinoxalinecarboxylic acid, 2-(acetyloxy)ethyl ester, 1,4-dioxide To a solution of 251.6 g. (1.85 moles) of benzofuroxan and 348 g. (1.85 moles) of 2-acetoxyethyl acetoacetate in 750 ml. of dimethylformamide at 5065C. is added 370 ml. of a 1 M solution of sodium ethoxide in ethanol. The reaction mixture is stirred at 50C. for 4 hours after which it is cooled, concentrated to one-half its volume and filtered. The resulting crude product is dissolved in chloroform, and the chloroform solution washed alternately with water, salt solution and water. The organic layer is separated, dried over anhydrous sodium sulfate and concentrated in vacuo to dryness. The residual solid is triturated with ether, filtered and dried, 179 g., m.p. 131133C., with decomposition.

Anal. Calcd. for C H O N C, 54.9; H, 4.6; N, 9.2.

Found: C, 54.7; H, 4.7; N, 9.1.

EXAMPLE II 3-Methyl-6- and 7-chloro-2-quinoxalinecarboxylic Acid. 2-(acetyloxy)ethyl Ester, l,4-dioxide To 255 g. (0.15 mole) of -chlorobenzofuroxan and 28.2 g. (0.15 mole) of 2-acetoxyethyl acetoacetate in 75 ml. of dimethylformamide at 50C. is added dropwise ml. of ethanol containing 345 mg. of reacted sodium metal. The resulting reaction mixture is allowed to stir at 50C. for an additional 4'"'hoursfollowed by the removal of the solvent under reduced pressure. The residual dark oil is dissolved in a minimum amount of chloroform and placed on a column containing 250 ml. of silica in ethyl acetate. The first 300 ml. of the eluate, benzene, is discarded and the subsequent 1.4 liters collected and concentrated to an oilpTrituration with ether results in crystallization of the desired product, which is purified by recrystallization from ethyl ether hexane, 3.74 g.

EXAMPLE Ill The procedures of Examples 1 and ll are repeated, using equivalent amounts of the requisite starting benzofuroxans and acetoacetates, to produce the following -Continued The procedures of Examples I and]! are again repeated, using the appropriately substituted benzofuroxan and acetoacetates to provide the following Continued Continued 0 N I I CO2AZ N X COZAZ X cH3 N cHa l N O l x A z A o CH CH X Z OCH CH CH CH,(CH C0 CH3 -C C 2 I ocH. CH CH cu co 1 OCH CH CH HCO CH CH ?H(CH2)2 z CH CHCH CH HC C H CH CH 3 3 fWCHz): ocH. CHCH CH HCO OCH l CH CH -C a a a I OCH; CH2CH2 CH3( Hz)2 2- C2145 OCH CH,CH,- CH CO, OCH, CH C(CH CH,- HCO CHzCHP' OCH CH,C(CH CH, CH3CO2 OCH .-CH(CH,) cu co OCH; CH2CH(C2H5)CH2 CH3CO2- 85:1 gfizgggzgs) gz a( z)z 2 z s a )C HCO OCH CH CH H O ocl-i (cllflhd 5 2 cn c o a 2): C 1 2r srsr- 2 z- CH z- CF: CH:(CH2)2COT OCH CH(CH2)2 (C14,),Cl-lc CF; CHCH2 CH3(CH2)2CO2- H I 2 5 CH:I 30 OCH: CH C(CH;) CH HCO2- f" CHaCOP 32: zrzsars: 22 22? 3 2 2 5) 2 3 2 H OCH; CH2CH(CH5)CH2 CH;(CH)CO= CF; CHCH2 (CH3)ZCHCOZ 5z s) 2 3 2 I! 2 CF CH CO2 CF; CH CH HCO 2 4 3 CF: CH2CH2 CH3CO2- CF: CH CH(CH,) CH3CO2 CF; CH2CH2 a( z)z z c|= CH-CH,- CH;,(CH=)=CO, CH3 l 0 CH 1 40 CF; CH-CH1- CH,CO

CH3 =AZ H (CHa)2CHCO X 3 a (CH2)4-. CH3CO2 CF; CH CH(CH 01.00,- l CH;

x A Z EXAMPLE V H CH CH HCO 3Methyl-2-quinoxalmecarboxylic acid, H CHaCH2CoZ 2-hydroxyethyl ester, 1,4-dioxide H CH CH CH (CH )CO j i a i To 100 ml. of a 12N hydrochloric acid solution is z)a Ll added 0. H 2,: a 2 l g l6 mole) of 3 methyl 2 H (CHz)s HCO2 qumoxa mecarboxyllc acid, 2-(acetyloxy)ethyl ester, H (|:HCHZ' z- 1,4-d1ox1de, and the resulting reaction mixture allowed CH3 to stir at room temperature for 1 hour. Two hundred H milliliters of water and 200 ml. of chloroform are added CH: to the mixture which is then cooled and adjusted to pH a l z a 2 2 5, using a 10% sodium hydroxide solution. The mixture CHa is further extracted (4 X 100 ml.) with chloroform, and (CH:) HCO 3 z 3 Z the combined chloroform layers dried over anhydrous CH3 (CHH)2CHCO2 sodium sulfate and concentrated to dryness under re- C (|:H(CH2)2' (CHQZCHCOF- duced pressure. The resulting residue is recrystallized CH3 from chloroform hexane to provide the pure product 3 ?H(CH2)2 C zas a yellow solid, 33.3 g., m.p. l46148C.

CHa Anal. Calcd. for C H O N C, 54.5; H, 4.6; N, C 3 C 2CH- C JC 2 10.6. Found: C, 54.2; H, 4.5; N, 10.7.

EXAMPLE \(1 The procedure of Example V is repeated, using the appropriately substituted quinoxaline-di-N-oxides, to provide the following analogs:

3-Methy]-2-quinoxalinecarboxylic acid, Z-aminoethyl ester, 1,4-dioxide hydrochloride EXAMPLE VIII The procedure of Example VIl is repeated, using the appropriately substituted starting materials, to provide the following congeners:

EXAMPLE IX 3-Methyl-2-quinoxalinecarboxylic acid, 2-(N-methylamino)-ethyl ester, 1,4-dioxide hydrochloride To 25 ml. of ethanol containing 3.8 g. (0.01 mole) of 3-methyl-Z-quinoxalinecarboxylic acid, 2-(N-tbutyloxycarbonylamino)ethyl ester, 1,4-dioxide is added 1.4 ml. of a M hydrochloric acid solution. The reaction mixture is heated in a water bath to 30C. for a period of minutes after which, it is concentrated to dryness under reduced pressure. The hydrochloride salt of the desired product is triturated several times with ethyl acetate and filtered.

EXAMPLE X The procedure of Example IX is repeated, employing the requisite starting materials, to provide the following products:

The procedure of Example IX is again repeated, using the appropriate starting materials, to produce the following congeners:

3-Methyl-2-quinoxalinecarboxylic acid, 2-(dimethylamino)ethyl ester, l,4-dioxide hydrochloride To a solution of 204 g. (1.5 moles) of benzofuroxan and 259 g. (1.5 moles) of 2-(dimethylamino)ethyl acetoacetate in 600 ml. of dry dimethylformamide is added over a 15 minute period ml. of l N sodium ethoxide in ethanol (0.15 mole). The resulting exothermic reaction raises the reaction temperature to 55C. where it is maintained for 3 hours after the reaction subsides. The solvent is removed under reduced pressure, 1 liter of water is added and the product extracted with (5 X 200 ml.) methylene chloride. The combined methylene chloride extracts are extracted with (3 X 200 ml.) 0.5 N hydrochloric acid and the combined aqueous acid layers basified with potassium carbonate and reextracted with (4 X 200 ml.) methylene chloride. The methylene chloride extracts are combined, dried over anhydrous sodium sulfate and concentrated in vacuo to an oil. The residual oil is dissolved in 500 ml. of methanolic hydrogen chloride, and the resulting solution cooled. The desired product crystallizes as the hydrochloride salt, which is filtered, washed with ethyl acetate and dried, 138 g., mp. 192C. with decomposition.

Anal. Calcd. for C H O N Cl: C, 51.3; H, 5.5; N, 12.8.

Found: C, 51.2; H, 5.7; N, 12.8.

EXAMPLE XIII The procedure of Example XII is repeated, using ap- EXAMPLE xiv The experiment conditions" of Example XII are repeated, using the requisite starting (materials, to produce the following compounds:

3-Methyl 2-quinoxalinecarboxylic acid, 2-aminoethyl ester, 1,4-dioxide hydrobromide 7 To a solution of 3.0 g. (0.01 mole) of 3-methyl-2- quinoxalinecarboxylic acid, 2-aminoethy1 ester, 1,4- dioxide hydrochloride dissolved in 10 ml. of water at 0C. is added 1.25 g. (0.005 mole) of silver oxide. The

suspension is allowed to stir briefly in the cold followed by centrifugation of the solid suspension. The supernatent solution containing the free base is treated with 1.7

'g. (0.01 mole) of a 48% hydrobromic acid and the resultingsolution concentrated to dryness in vacuo. The

resulting hydrobromide salt is triturated with ethyl acetate and suction filtered.

EXAMPLE XVI Employing the procedure of Example XV the hydrochloride salts of the products of Example Vlll are converted to their respective hydrobromide, sulfate and phosphate salts.

EXAMPLE XVII Employing the aforementioned two-fold serial dilution technique, the in vitro activity of some of the products of the instant invention against Staphylococcus aureus' and Escherechia coli are presented. Benzylpenicillin (K Salt) when tested gave MlC (Minimal Inhibitoi'y Concentration) values of 0.156 and vs. S. aurus and E. coli, respectively.

EXAMPLE XIX The efficacy of the herein described compounds in o protecting against a systemic challengeinfection of Sall monella chulerasuis var. kunzendorf in swine is demonstrated by the following experiment. Young, 6-8 week old pigs are conditioned for fourteen 'days in isolation X rooms and maintained during the entire study on a basal ration consisting of ground yellow corn (78.4%), 4 soybean meal (15%), alfalfa meal (2%), meat bone l scraps (2.5%), limestone (0.4%), dicalcium phosphate 0 (0.65%), iodized salt (0.5%), Vitamin pre-mix PPM No. 5 (0.5%), quadruple delamix (0.05%) and zinc carbonate (7.8 g./1OO lbs. mix). All the pigs, which are divided into groups of six, are inoculated on day 0 with 4 ml. (approximately 2.0 X 10 organisms) of the stock inoculating suspension. Treatment with the quinoxaline-di-N-oxides of the present invention is carried out on day 0 and day 1 by intramuscular injection at 12 X R4 A Z hour intervals at doses of 2.5 and 5 mg./kg. On day 10 H CH: (CH2)2- C H CO the percent mortality In each group is calculated. The H CH3 (CH2)4 NC H CO 3 following results are obtained: H car-l, (CH (343C021 6 2 H C3H1 (CH CH(CH,-i) CH3CO2 E Eg:2)a 4ClC H CO 3CHOCHCO- Medication Mortality F CZ": v (CH:); c q c d z I C] CH3 I (CH2)2 C5H5CO2- 'i ggfiif med'cded (P'dcebo 83 gi l' s c i c(cna)zcuz CH;C0= B-MethyLZ-quinoxalinecarboxylic acid, Br CZHZ ;FCC6HlCO 2-(acetyloxy)ethyl ester, l,4dioxide Br C H (CH 2.5 mgJkg. x 4 67 CH 2 a 5 0 g g X 4 7 a 2 s 2)z 4BIC5H4CO2 CH3 rs 1 2)z CH3CO2 C 3 CQ 1 (CH)2 4 ClC.,H,Co OCH: a -'-(CH-;) I C5H5CO OCH; C2H5 -(CHz)z a 2 EXAMPLE XX z s H)= csnsco OCH :1 a 7 (CH2)5 CH3CO2 Employing the previously described procedure for 5:: g:z;z 5H=C02 determining growth promotion in animals, the follow- C ii, (CH:): 4Cggggmcor mg qumoxalme-dl-N-oxides were tested at g./ton of CF: C2H5 -(C z)z C HSCOP feed in swine for a period of 28 days and provided the jgmcHficHzcmcH CH=COP following results: 3

CO AZ X I Growth X A Z Weight Gain lndex* Over Control H -(CH,), N(CH,).HCI 129 29 0 ll H (CH;); O CCH, 153 53 H -(CH,) NH,.HC| I39 39 control=l00 EXAMPLE XXI The experimental procedure of Example I is repeated, using the appropriate B-ketoeste'rs and benzofuroxans, to provide the following compounds:

7 EXAMPLE XXll Employing the procedure of Example V and starting with the requisite quinoxaline-di-N-oxide, the following analogs are prepared:

EXAMPLE XXIII EXAMPLE XXIV The procedure of Example IX is again repeated, using the requisite starting material, to provide the following analogs:

EXAMPLE XXV The procedure of Example XII is repeated, using the appropriate starting materials, to provide the following --Continued EXAMPLE XXVI 3-Methyl-2-quinoxalinecarboxylic acid, 2-(acetoxy)ethyl ester, l,4-dioxide via Scheme I EXAMPLE xxvu 1 3Methyl-2-quinoxalinecarboxylic acid, 2-(dimethylamino)ethyl ester, 1,4-dioxide via Scheme To 250 ml. of toluene is added 27.2 g. (0.2 mole) of 3-methyl-2-quinoxalinecarboxylic acid, .methyl ester, l,4-dioxide, 17.8 g. (0.2 mole) of dimethylaminoethanol and 37.8 g. (0.22 mole) of .p-toluenesulfonic acid and the resulting mixture is heated to reflux. During the reaction period the reflux condenser is removed and the tolueneis allowed to boil off, removing methanol, the by-product of the reaction. When the reaction is complete the solvent is removed vacuo and the residue partitioned between water and chloroform. The water layer is then separated and made jslightly basic by the careful addition of a sodium hydroxide solution. The basic solution is then extracted with methylene chloride, dried over sodium sulfate vand the solvent removed under reduced pressure. Addition of methanolic hydrogen chloride to the residue provides the hydrochloride of the desired product, identical to that prepared in Example Xll.

EXAMPLE xxvm 3-Methyl-2-quinoxalinecarboxylic acid, 2-(acetoxy)ethyl ester, 1,4-dioxide via Scheme III A.mixture of 20.1 g. (0.1' mole) of 2 -cyano- 3methylquinoxaline-l,4-di-N-oxide, 37.6 g. (0.2 mole) of 2-acetoxyethanol in 200 ml. of toluene is treated with 10ml. of 12 N hydrochloric acid solution and the mixture heated at steam bath temperatures until the reaction is complete. The excess alkanol, solvent and water are removed in vacuo, and the residue is triturated several times with ether. The product isolated isidentical to that prepared in Examples I and XXVI.

EXAMPLE XXIX The procedures of Examples XXVI, XXVII and XXVIII representing Schemes I, II and III, respectively, are repeated, using the appropriate starting reagents and reaction conditions, to provide the following quinoxaline-di-N-oxides:

EXAMPLE XXX 3-Methyl-2-quinoxalinecarboxylic acid, 2-(4-c -arboxypropionyloxy )ethyl ester, l,4-dioxide A mixture of 26.4 g; 0.1 mole) of 3-methyl-2- quinoxalinecarboxylic acid, 2-hydroxyethyl ester, 1,4-

, reduced pressure. The residual product is induced to crystallize from methanol, 24.7 g., m.p. l67 C. Anal.Calcd. for C ll O N z C, 52.8; H, 4.4; N, 7.7. Found: C, 52.9; H, 4.6; N, 7.6.

By substitution of glutaric anhydride for -succinic anhydride in the above example 3-methyl-2- Found: C, 53.7; H, 4.8; N, 7.4. 5

EXAMPLE xxx! Following the general procedure of Example XXX and starting with the appropriate hydroxyalkyl ester of 3-methyl-2-quinoxalinecarboxylic acid, 1 ,4-dioxide from Example VI and requisite anhydride or malonyl or oxalyl chloride, the following products are prepared:

3-methyl-2-quiinoxalinecarboxylic acid, 2- (propionyloxy)ethyl ester, 1,4-dioxide, m.p. 95 C.; 3- methyl-2-quinoxalinecarboxylic acid, 2- (butyryloxy)ethyl ester, l,4-dioxide, m.p. 6869" C.; 3-methyl-2-quinoxalinecarboxylic acid, 2- (valeryloxy)ethyl ester, 1,4-dioxide, m.p. 5660 C.; 3-methyl-2-quinoxalinecarboxylic acid, 2- (caproyloxy)ethyl ester, 1,4-dixide, m.p. 7072 C.; 3-methyl-2-quinoxalinecarboxylic acid, 2- (enanthyloxy)ethyl ester, l,4-dioxide, m.p. 6870 C.; 3-methyl-2-quinoxalinecarboxylic acid, 2- (capryloxy)ethyl ester, 1,4-dioxide, m.p. 6062 C.; 3-methyl-2-quinoxalinecarboxylic acid, 2- (chloroacetoxy)ethyl ester, 1,4-dioxide, m.p. l l5l 17 C.; 3-methyl-2-quinoxalinecarboxylic acid, 2-[(ethoxycarbonyl)oxy]ethyl ester, 1,4-dioxide; and 3-methyl-2-quinoxalinecarboxylic acid, 2- [(isobutoxycarbonyl)oxy]-ethyl 1,4-dioxide, m.p. 7273 C.

EXAMPLE XXXlll The procedure of Example XXX]! is repeated, employing the requisite acid halides and hydroxyalkyl esester,

ters of 3-methyl-2-quinoxalinecarboxylic acid, l,4-

dioxide of Example VI, to provide the following analogs:

CO2AZ 't CH, 9 20 H (CH) O CCH CO H H (CH2)2 OZC(CHZ)4CO2H H 2)z 02C(CH2)CO2H H z):|- O2C(CH2)2CO2H H CH(CH3)CH2 O2CCO2H F (CH2)2 -O2C(CH)5CO2H F -CH(C H )CH O2CCH2CO,H F (CH2): OC(CH2),CO2H F 2)a O2C(CH2)5 C02" Cl CH(CH )CH O CCH,C(CH CH CO,H Cl -(CH2) -0,CCH,C CH, CH CO H Cl (CH O,C(CH)4CO2H Cl CH(C;,H1)CH, O C(CH)aCO H Br (CH2)4 O2CCH(CH -,)CO2H Br -CH,C(CH CH, -0,c(CH,) C0,H Br CH2C(CH3)gCHg 2( 2)G z CH: (CH:)2 O2CCOZH CH; (CH,); O2CCH2CO,H CH, -CH(CH 1)CHZCH(CH- O2CCH2CO2H CH CH(CH;, O CCH- CH(C H )CH,C0 H

)CH2CH(CH;|) OCH; (CH) O2CCH2CH(C2H5)CH,CO,H OCH: (CH2)2 O- IC(CHZ)4COH OCHQ (CH:) O2C(CH=);COH OCH; CH2C(CH,)=CH2 O,C(CH;)|CO,H a (C 2)5- O C(CH,),CO,H CF; -(CH)4 O2CCH(CH,)CO2H CF: (CH2)4 OCCO2H F. -(CH2) -o,cco,H CF; (CH2)2 -O C(CH CO,H E: z)z z 2)2 2 EXAMPLE XXXll 3-Methyl-2-quinoxalinecarboxylic acid, 2-(octanoyloxy)ethyl ester, 1,4-dioxide To a solution of 26.4 g. (0.1 mole) of 3-methyl-2- quinoxalinecarboxylic acid, 2-hydroxyethyl ester, 1,4- dioxide and 30.3 g. (0.3 mole) of triethyl amine in 200 ml. of methylene chloride and cooled to 0 C., is added dropwise 32.5 g. (0.2 mole) of octanoyl chloride in 50 ml. of the same solvent. After stirring at 0 C. for 1 hour the reaction mixture is allowed to warm to room temperature and is subsequently washed with a saturated sodium bicarbonate solution and then with 6N hydrochloric acid. The organic layer is separated, dried over magnesium sulfate and evaporated in vacuo to an amber oil which is induced to crystallize with ether petroleum ether, 25.7 g., m.p. 6264 C. 65

By replacing octanoyl chloride with the appropriate acid chloride, the following compounds are similarly prepared:

EXAMPLE XXXIV 3-Methyl-2-quinoxalinecarboxylic acid, 2-( N-ethylcarbamyloxy)-ethyl ester, 1,4-dioxide EXAMPLE XXXV Starting with the appropriate 3-methyl-2- quinoxalinecarboxylic acid, hydroxyalkyl ester, 1,4- dioxide and alkyl isocyanate, and repeating the procedure of Example XXXIV, the following quinoxaline- 1,4-dioxides are synthesized:

EXAMPLE XXXVI 3-Methyl-2-quinoxalinecarboxylic acid, 2-(dimethylaminoacetoxy)ethyl ester, 1,4-dioxide To a solution of 3.4 g. (0.01 mole) of 3-methyl-2- quinoxalinecarboxylic acid, 2-(chloroacetoxy)ethyl ester, l,4-dioxide in 40 ml. of methylene chloride is added, with cooling, 1.0 g. (0.022 mole) of dimethyl amine in 10 ml. of the same solvent. The reaction mixture is allowed to stir for several hours at room temperature and is then heated to 40-50 C. for an additional hour. The mixture-is poured into water and the organic phase separated, dried over sodium sulfate and concentrated to dryness. The crude product is purified by recrystallization-from chloroform ether.

A small portion of the free base is dissolved in chloroform and sufficient hydrogen chloride dissolved in ethyl acetate added to precipitate the hydrochloride salt.

EXAMPLE XXXVI] Employing the procedure of Example XXXVI, and starting with the appropriate secondary, dialkylamine and bromo or chloro compound from Example XXXIII, the following tertiary amines are prepared: 

1. A METHOD FOR PROMOTING GROWTH AND IMPROVING FEED EFFICIENCY IN SWINE WHICH COMPRISES ORALLY OR PATENTERALLY ADMINISTERING TO SAID SWINE A DOSAGE AMOUNT OF FROM ABOUT 0.1 MG/KG TO ABOUT 100MG/KG OF BODY WEIGHT PER DAY OVER A MAJOR PORTION OF THE SWINES ACTIVE GROWTH PERIOD A COMPOUND OF THE FORMULA 